Sialyl-Tn (STn) is a tumor-associated carbohydrate antigen overexpressed in various carcinomas. To obtain its expression, murine carcinoma cells were transfected with the cDNA encoding ST6GalNAc I, a glycosyltransferase that acts exclusively on O-glycans. Overexpression of this enzyme led to the expected expression of cell surface STn epitopes. Surprisingly, the transfectants (STn+ cells) presented dramatic morphological changes and altered behavior. These STn+ cells lost the epithelial appearance of parental cells, became larger, more elongated and presented disorganized actin stress fibers. Additionally, their proliferation was impaired and their ability to migrate on fibronectin and hyaluronic acid was severely reduced. By contrast their adhesion on fibronectin remained unchanged. The major glycoprotein carrying the STn epitope was shown to be the integrin beta1 subunit. Anti-STn antibodies could restore migration of STn+ cells on fibronectin. A constitutively active permeant form of RhoA (TAT-RhoA(Val-14)) also restored motility on fibronectin of STn+ cells as well as a parental STn-cellular phenotype. These observations indicate that overexpression of ST6GalNAc I leads to a major change of the O-glycosylation of the integrin beta1 chain which in turn impairs the integrin-mediated signalling and leads to major alterations in morphology and cell behavior.